Alkaline phosphatases are commonly used in routine biochemical procedures to remove phosphate groups from the termini of nucleic acid molecules. For example, calf intestinal alkaline phosphatase is a heat labile enzyme which is used to remove such phosphate groups, and then is inactivated by exposure to a high temperature. This thermal instability is advantageous because the alkaline phosphatase need not be removed from the reaction mixture prior to subsequent manipulations.
Alkaline phosphatase is also used as a non-radioactive marker for the detection of specific protein or DNA targets. It is conjugated to proteins or DNA oligonucleotides to aid in detection of such targets. Enzyme thermostability is desirable for use in labelling DNA molecules, especially those larger than 50 bp, and may be useful in other applications for increased stability of the enzyme during storage.
The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
Alkaline phosphatases from various thermophilic and other organisms are known: Yeh and Trela (1976) "Purification and Characterization of a Repressible Alkaline Phosphatase from Thermus aquaticus" J. Biol. Chem. 251:3134-3139; Hartog and Daniel (1992) "An Alkaline Phosphatase from Thermus sp Strain Rt41A" Int. J. Biochem. 24:1657-1660; Schaffel and Hulett (1978) "Alkaline Phosphatase from Bacillus licheniformis" Biochimica et Biophysica Acta 526:457-467; Hulett-Cowling and Campbell (1971) "Purification and Properties of an Alkaline Phosphatase of Bacillus licheniformis" Biochem. 10: 1364-1371.